The mitochondrion represents a target of reactive oxygen stress and mitochondrial DNA appears to be an early and sensitive marker of this stress. Many human diseases are associated with reactive oxygen, including cancer, heart disease and neurodegenerative diseases. Mitochondria are essential organelles for generating ATP during oxidative phosphorylation. The mitochondrial DNA encodes 13 polypeptides, eleven are involved in electron transport and two serve as subunits of ATP synthase. Damage to mitochondrial DNA is repaired, but prolonged oxidant treatment results in persistent mtDNA damage, loss of mitochondrial function, increase in p21Waf1/CIP, and apoptosis. These observations suggest that mitochondrial injury, specifically DNA damage, is important for reactive oxygen- induced toxicity. We are testing the hypothesis that reactive oxygen species (ROS) generated in the mitochondria result in mtDNA damage, which in turn causes the release of more ROS (superoxide, hydrogen peroxide, and the hydroxyl radical) that lead to further mitochondrial decline and many degenerative diseases associated with aging. In collaboration with Dr. Steve Kleeberger, hyperoxia is being used to look at the induction of mitochondrial DNA damage. [unreadable] [unreadable] We are analyzing the effects of hydrogen peroxide on cells immoratilized by expressing the protein component of telomerase, hTERT. [unreadable] Telomerase is often re-activated in human cancers and widely used to immortalize cells in culture. Besides the maintenance of telomeres, telomerase has been implicated in cell proliferation, genomic instability and apoptosis. Here we show that hTERT is targeted to the mitochondria by an N-terminal leader sequence, and mitochondrial extracts contain telomerase activity. In seven different human cell lines, mitochondrial telomerase increases hydrogen peroxide-mediated mitochondrial DNA damage. hTERT expression did not alter the rate of hydrogen peroxide breakdown or endogenous cellular levels. Since the damaging effects of hydrogen peroxide are mediated by divalent metal ions (Fenton chemistry), we examined the levels of bioavailable metals. In all cases, higher levels of chelatable metals were found in hTERT-expressing cells. These results suggest that mitochondrial telomerase sensitizes cells to oxidative stress, which can lead to apoptotic cell death, and imply a novel function of telomerase in mitochondrial DNA transactions. [unreadable] [unreadable] We are also looking at another system in which we have in collaboration with Christi Walter, have engineered a mitochondrially-targeted EcoRI construct that is under the control of doxycycline, such that withdraw of DOX leads to its expression in both tissue culture cells and in mice.